Summary of the research project proposed by Dr. Robert L. Raffai, PhD: Diabetes is associated with a 2- to 4-fold increase in atherosclerosis-related cardiovascular disease including peripheral arterial disease (PAD). Despite this association, little is known about the mechanisms by which diabetic hyperglycemia can accelerate atherosclerosis and complication of PAD that include limb loss and premature death. Moreover, whether hyperglycemia can impair the regression of atherosclerosis is not known. Unfortunately, studies of existing mouse models of atherosclerosis were often marred by the recently recognized effect by which hyperglycemia increases plasma cholesterol levels in diabetic mice. Studies in which hyperglycemia was found to accelerate atherosclerosis also reported increased plasma cholesterol in diabetic mice. Moreover, the lack of suitable mouse models of atherosclerosis regression have hampered studies designed to address the effects of diabetic hyperglycemia on atherosclerosis regression, a promising clinical treatment strategy for cardiovascular disease. We propose to overcome these limitations by studying our newly developed mouse model of spontaneous and reversible dyslipidemia and atherosclerosis called hypomorphic apolipoprotein E mice deficient in the low density lipoprotein receptor (Apoeh/hLdlr-/-Mx1-Cre mice). When fed a low-fat chow diet, these mice, while moderately dyslipidemic, are resistant to further hyperglycemia-induced hypercholesterolemia. Moreover, they develop occlusive peripheral atherosclerosis by 12 to 14 months of age, predisposing to critical limb ischemia and premature death while on a chow diet. Remarkably, their dyslipidemia can be permanently lowered by conditional gene repair of the hypomorphic Apoe allele mediated by inducible Cre activation, which results in atherosclerosis regression within 2 weeks. In Aim 1, we will study hyperglycemic and normoglycemic Apoeh/hLdlr-/-Mx1-Cre mice to assess the extent and frequency of occlusive peripheral atherosclerosis and symptomatic forms of PAD in 12 month old mice. We will also test the hypothesis that in the absence of added dyslipidemia, hyperglycemia enhances systemic inflammation and the pro-inflammatory state of vascular cells in peripheral arteries. We will then test the hypothesis that enhanced vascular inflammation augments monocyte recruitment in peripheral arteries, and that hyperglycemia raises intracellular free cholesterol levels, thereby promoting the unfolded protein response and premature apoptosis of macrophage foam cells. Lastly, we will test the hypothesis that high density lipoproteins isolated from hyperglycemic Apoeh/hLdlr-/-Mx1-Cre mice will display a reduced ability to promote lipid elimination and suppress inflammation, and that low density lipoproteins will have opposite effects. The rapid reversal of dyslipidemia and atherosclerosis in Apoeh/hLdlr-/-Mx1-Cre mice provides a powerful new approach to address mechanisms associated with atherosclerosis regression. Thus, in Aim 2, we will test the hypothesis that hyperglycemia impairs transcriptional reprogramming of vascular cells and thereby the removal of arterial lipid, the egress of macrophages and the accumulation of collagen in response to lipid lowering. Next, we will explore the utility of therapeutic insulin treatment to enhance atherosclerosis regression in hyperglycemic Apoeh/hLdlr-/-Mx1-Cre mice. Our long-term goals are to identify mechanisms that could serve as therapeutic targets to delay the progression and accelerate the regression of atherosclerosis as treatments for PAD in diabetic individuals.